Nitric oxide (NO) is a pluripotent physiological mediator playing a key role in the control of a large number of biological functions. In addition to these essential biological effects NO has also been shown to be mutagenic in both prokaryotypic and eukaryotic systems. The objective of this project is to assess where the balance lies between these two seemingly paradoxical effects of NO on the carcinogenic process. To study the effects of NO in vivo transgenic mice overexpressing NOS are being generated. The chronic toxicity of NO observed in cells precludes the use of constitutively active promoters so two regulatable elements are being employed: a 552 bp fragement of the mouse beta-casein promoter, which is selectively active in the lactating mammary gland, and a 621 bp fragment of the rat phosphoenolpyruvate carboxykinase (PEPCK) promoter, which is expressed in a number of tissues, including the liver, and can be regulated by hormonal and dietary manipulation. A casein-NOS chimeric gene has been constructed and has been found to be active in vitro in Chinese hamster ovary (CHO) cells when co-expressed with the long form of the mouse prolactin receptor. The expression from the casein/NOS construct can be hormonally modulated in a manner similar to that in published reports using the native beta-casein gene or casein promoter/reporter constructs. The casein-NOS construct has been microinjected into C57B16 x CBA mouse embryos at the two-cell stage and pups that carry and transmit the transgene, as assessed by discriminatory polymerase chain reaction (PCR), have been obtained. A PEPCK-NOS chimeric gene has also been constructed and is being tested in cell lines and primary hepatocytes to ensure that NOS expression occurs in a hormone dependent manner. This construct will then be used for the generation of transgenic mouse lines.